In the past, the following methods have been known as a method for preparing .alpha.-hydroxy acid [II] by using .alpha.-hydroxynitrile [I] as the starting material and by means of using microorganisms.
(1) A method for preparing lactic acid, glycolic acid, etc. by using a microorganism, such as Bacillus spp., Bacterizium spp., Micrococcus spp. and Brevibacterium spp., which is disclosed in Japanese Patent Publication No. Sho 58-15120. PA0 (2) A method for preparing lactic acid, glycolic acid and 2-hydroxyisobutyric acid by using a microorganism belonging to Corynebacterium spp., which is disclosed in Japanese Patent Laid-open No. Sho 61-56086. PA0 (3) A method for preparing lactic acid, 2-hydroxyisobutyric acid, 2-hydroxy-2-hydroxyphenyl propionic acid and mandelic acid by using a microorganism, such as Pseudomonas spp., Arthrobacter spp., Aspergillus spp., Penicillium spp., Cocryoboros spp. and Fusarium spp., which is disclosed in Japanese Patent Laid-open No. Sho 63-222696. PA0 (4) A method for preparing 2-hydroxy-3,3-dimethyl-4-butyrolactone by using a microorganism, such as Arthrobacter spp., Aspergillus spp., Bacillus spp., Bacterizium spp., Brevibacterium spp., Cocryoboros spp., Corynebacterium spp., Micrococcus spp., Nocardia spp., Penicillium spp., Pseudomonas spp. and Fusarium spp., which is disclosed in Japanese Patent Laid-open No. Sho 64-10996. PA0 (5) A method for preparing 2-hydroxyisobutyric acid by using a microorganism, such as Rhodococcus spp., Pseudomonas spp., Arthrobacter spp. and Brevibacterium spp., which is disclosed in Japanese Patent Laid-open No. Hei 4-40897. PA0 (6) A method for preparing .alpha.-hydroxy-4-methylthiobutyric acid by using a microorganism, such as Caseobater spp., Pseudomonas spp., Alcaligenes spp., Corynebacterium spp., Brevibacterium spp., Nocardia spp., Rhodococcus spp. and Arthrobacter spp., which is disclosed in Japanese Patent Laid-open No. Hei 4-40898. PA0 (7) A method for preparing 4-methylthiobutyric acid by using a microorganism, such as Alcaligenes spp., Rhodococcus spp. and Goldona spp., which is disclosed in WO 96/09403. PA0 (8) A method for preparing .alpha.-hydroxy-4-methylthiobutyric acid by using a microorganism, such as Pantoea spp., Micrococcus spp. and Bacterizium spp., which is disclosed in Japanese Patent Laid-open No. Hei 8-173175. PA0 Deposition No.: FERM BP-5829 (Transferred from Bikouken Microorganism PA0 Deposition No. P-15424) PA0 Date of Deposition: Domestic deposition has been made on Feb. 6, 1996 and international deposition has been made on Feb. 20, 1997. PA0 Place of Deposition: No. 1-3, Higashi 1-chome, Tsukuba-shi, Ibaragi, Japan PA0 Organization for Deposition: Institute for Biotechnology and Industrial Technology, Industrial Technology Academy, Ministry of trade and Industries PA0 Shape: Polymorphous bacillus PA0 Gram's stain property: Positive PA0 Rod-coccus cycle: Positive PA0 Spores formation: Negative PA0 Mobility: Negative PA0 Diamino acid in Cell Wall: Lysine PA0 Oxigen requirement: Aerobic PA0 Oxidase formation: Negative PA0 Catalase formation: Positive PA0 DNA decomposition: Positive PA0 Liquefaction of Gelatin: Positive PA0 Starch decomposition: Positive PA0 Casein decomposition: Positive PA0 Vitamins requirement: Negative PA0 Glycolyl test: Negative (Acetyl-type) PA0 Quinone type: MK-9 (H2) PA0 Sugar composition of Cell Wall: Galactose + Glucose +
However, the methods for preparing .alpha.-hydroxy acid as mentioned above are not always recognized as satisfactory one which can produce and accumulate an objective substance at a high concentration. For example, in case of lactic acid, only 9.8% by weight accumulation thereof is recognized when one of Corynebacterium spp. is used as the microorganism (Japanese Patent Laid-open No. Sho 61-56086), only 10% by weight accumulation thereof is recognized when one of Pseudomonas spp. is used (Japanese Patent Laid-open No. Sho 63-222696) and only 0.15% by weight accumulation thereof is recognized when one of Arthrobacter spp. is used (Japanese Patent Laid-open No. Sho 63-222696). Whereas, the accumulation of .alpha.-hydroxyisobutyric acid by using one of Pseudomonas spp. is found to be as much as 0.8% by weight (Japanese Patent Laid-open No. Sho 63-222696), the accumulated amount of .alpha.-hydroxy-4-methylthiobutyric acid is found to be 188 mM (2.8% by weight) by using one of Caseobater spp. (Japanese Patent Laid-open No. Hei 4-40898), 55 mM (0.8% by weight) by using one of Arthrobacter spp. (Japanese Patent Laid-open No. Hei 4-40898) and 940 mM (14% by weight) by using one of Alcaligenes spp. (WO 96/09403), respectively.
As a reason for the low accumulation concentration of such products as described above, it is considered that some enzymatic activity relating thereto might be inhibited in the presence of cyanic acid (Agricultural Biological Chemistry, Vol.46, page 1165, 1982) which is generated in the partial dissociation of .alpha.-hydroxy nitrile in water together with the corresponding aldehyde or ketone (Chemical Reviews, Vol.42, Page 189, 1948). Further, a possibility that the related-enzyme is inactivated in a short time with the dissociated-aldehyde has been also pointed out. As a solution to prevent such inactivation of the enzyme, a method to add either acidic sulfite ions or dithionite ions (Japanese Patent Laid-open No. Hei 5-192189) and a method to add either phosphite ions or hypophosphite ions (Japanese Patent Laid-open No. Hei 7-213296) have been proposed. However, the concentration of .alpha.-hydroxy acid produced and accumulated is not so high even by using such additives as described above.
In general, when accumulated-concentration of a product remains low, it is well known to the specialists in the art that the installation for such manufacturing tend to be complex and large. Therefore, there has been difficulty in the efficiency to manufacture .alpha.-hydroxy acid in an industrial scale according to the methods as described above. The present invention is to provide a method to accumulate .alpha.-hydroxy acid at a high concentration level by means of using microorganisms and to efficiently produce .alpha.-hydroxy acid.